Although a spectrum of analytical methods for small sample separation and purification have been developed, a number of problems, such as the slow speed of the separation process and the loss of sample volume, limits the quality of currently available methods. In the present invention, we describe the use of a pipette tip, which has one or more perforations, incisions, or holes in the tip, designed for sample filtration or separation. The perforations, holes or incisions are of such dimensions that fluids and smaller particles can pass through them while larger particles are retained in said pipette tip. The application of such a device is useful for performing sample separation and filtration with or without chromatography and other separation media because it eliminates the need to place a filter or secondary solid matrices into the tip to retain said chromatography and other separation media within the tip during sample preparation. Depending on the specifications of the chromatography material, selected molecules from the sample can be separated or purified by binding to, or by being entrapped in, said material. The bound molecules can later be eluted from said chromatography material by the use of different solvents.
Filters and secondary solid matrices are traditionally used in separation columns, such as pipette tips, to retain the solid matrix or column material in said pipette tips or columns, while letting fluids and desired sample components flow through. Currently available sample preparation pipette tips rely on filters and secondary solid support matrices to hold chromatography and separation media in the tip while permitting selective components of the sample to pass through the lower end of the tip. The filters used in such devices may be made of silica, polypropylene, nylon, polytetrafluoroethylene or any other inert materials that do not react with the elution solution. The pore size of the filter material is smaller than the particle size of the solid matrix or chromatography material placed above it so that the solid matrix or chromatography material is retained in the tip.
In pipette tips, or other separation/chromatography columns using filters, sample separation can be performed by centrifugation, gravitation, vacuum suction, pressure application or by syringe- or pipette-based sample delivery through the tip or column. Such methods are used for the separation and purification of small sample volumes of bio-molecules such as proteins, peptides and DNA. The solid matrix or column material can consist of any material such as gel-filtration, affinity, ion-exchange, reverse-phase, and silica or modified-silica materials.
When sample volumes in the micro liter range are separated using currently available pipette tips and other separation/chromatography columns, one of the commonly associated problems is sample loss during the separation process due to the retention of sample components on the filter or in the solid support matrix. Since the concentration of bio-molecules in micro volume samples is so small, the retention of molecules in the filter or secondary solid matrix can result in significant loss of the total sample volume. Also, since the volume of the filter or solid support matrix is often as large as the volume of the micro volume sample itself, the separation or chromatography process is adversely affected due to the large volume of the filter or solid support material through which the sample must pass during the separation process. The filter or solid support material may also absorb proteins or bio-molecules from the sample, resulting in lower than desired sample recovery.
Also, the filter material and solid matrix or column material may behave differently in different elution media, subsequently interfering with both the quality of the separation process and the volume of the sample retained. Furthermore, the presence of the filter material slows down the sample separation process because the sample is first passed through the column material and then the filter prior to the completion of sample separation. Additionally, the structure and design of many micro pipette tips, intended for micro-liter sample volumes, are not well suited to the addition of filters or secondary solid matrices into the tip.
The invention described and claimed herein comprises a pipette tip designed to perform filtration or sample separation on the basis of one or more perforations, incisions or holes performed on the tip for sample filtration or separation. The perforations, holes or incisions are of such dimensions that fluids and smaller particles can pass through them while larger particles are retained in said pipette tip. During the process of sample separation, performed by centrifugation, gravitation, vacuum suction, pressure application or by syringe-based sample delivery through the pipette tip described herein, the filter solid support matrix or column material is retained in said pipette tip while selected sample components pass through said perforation or incision.
The features of the present invention eliminate the need for filters or solid support matrices at the lower end of the pipette tip to support the chromatography material during sample separation. Thus, the present invention, as described herein, will result in higher sample recovery due to the absence of sample retention on a filter or solid support matrix. The present invention will also eliminate separation problems associated with filter materials and solid support matrices interacting differently with alternate elution media, thus improving the quality of sample separation. The present invention also leads to more rapid sample preparation since the sample does not flow through a filter or solid support matrices as part of the separation process. Filters and solid support matrices reduce the rate of sample flow.
The various features of novelty, which characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its advantages and objects, reference is made to the accompanying drawings and descriptive matter in which a preferred embodiment of the invention is illustrated.